Despite superior cytotoxicity against early-stage pancreatic tumors, gemcitabine is practically ineffective in late-stage tumors due to the invasive growth characteristics and apoptotic resistance of tumor cells. Evidence in literature suggest that epigenetic alterations (e.g. histone and DNA hypermethylation) may play vital roles in silencing tumor suppressor genes, allowing tumor cells to clonally expand and resist cytotoxicity. Our long-term goal is to improve the chemotherapeutic management of pancreatic cancer. The overall objective of this R03 grant application is to test the concept that the reactivation of epigenetically silenced tumor suppressor genes can augment chemosensitization in pancreatic cancer. Specifically, it is our central hypothesis that a mechanism-based structure optimization of a novel histone-demethylation agent, 3'deazaneplanocin-A (DZnep), in combination with gemcitabine will enact a superior cytotoxic response in pancreatic cancer cells. This hypothesis is based on our preliminary data that show transport-dependent augmentation of gemcitabine chemosensitization by DZnep in cultured pancreatic cancer cells. The rationale underlying the proposed research is that proof of synthetic demethylating agents improving gemcitabine chemosensitivity will provide an experimental basis to continue investigating the potential of epigenetic therapy for pancreatic cancer. The central hypothesis will be tested by pursuing three specific aims. Specific aim 1 will determine the cellular transport and activation mechanisms of DZnep in pancreatic cancer. The working hypothesis for aim 1 is that the demethylating and chemosensitizing abilities of hydrophilic DZnep will be rate-limited by a carrier-mediated transport process. This hypothesis is based on our preliminary data that show a significant reduction in DZnep-induced responses when nucleoside transport activity was inhibited. Specific aim 2 will generate and test a battery of DZnep acyl derivatives in vitro. The working hypothesis for aim 2 is that N4-substituted fatty acid amide derivatives will increase the lipophilicity of DZnep and allow it to bypass the transport requirement for intracellular activation. Our hypothesis is based on our preliminary studies showing the acyl prodrugs of another nucleoside (troxacitabine) bypassing the transport requirement. Specific aim 3 will determine the in vivo anti-cancer efficacies of DZnep prodrug-gemcitabine combinations. Our working hypothesis is that the combined treatment of a DZnep prodrug with gemcitabine in mice carrying pancreatic cancer xenografts will exhibit superior chemotherapeutic efficacy compared with gemcitabine treatment alone. This hypothesis is based on the extrapolation of preliminary in vitro results obtained with DZnep and gemcitabine combination schedules in cell culture. The contribution from these studies will be significant because successful demonstration of a superior epigenetic-chemotherapeutic regimen is likely to replace the less efficacious gemcitabine monotherapy practice. The proposed work is innovative because it addresses for the first time the possibility of a nucleoside-based epigenetic-chemotherapeutic therapy in pancreatic cancer.